Alzheimer's Disease and Frontotemporal Dementia: Methods and Protocols

290 Pages·2011·36 MB·English

by  Erik D. Roberson (auth.)

Alzheimer’s Disease
and Frontotemporal Dementia
Methods and Protocols
Edited by
Erik D. Roberson
Center for Neurodegeneration and Experimental Therapeutics,
Departments of Neurology and Neurobiology,
University of Alabama at Birmingham, AL, USA
Editor
Erik D. Roberson, M.D., Ph.D.
Center for Neurodegeneration and Experimental Therapeutics
Departments of Neurology and Neurobiology
University of Alabama at Birmingham, AL
USA
[email protected]
ISSN 1064-3745 e -ISSN 1940-6029
ISBN 978-1-60761-743-3 e-ISBN 978-1-60761-744-0
DOI 10.1007/978-1-60761-744-0
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2010938365
© Springer Science+Business Media, LLC 2011
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Preface
Alzheimer’s disease (AD) is the most common neurodegenerative disorder and one of the
most feared diseases due to the manner in which it robs its victims of their memories.
Frontotemporal dementia (FTD) is perhaps somewhat less well known among the public,
but it is also a prominent cause of dementia that produces devastating changes in personal-
ity and a decline in interpersonal interactions. The two conditions are often considered
siblings, for while they are distinct disorders targeting different brain regions and produc-
ing unique clinical symptoms, there is some overlap in their molecular neuropathology
(such as the presence of inclusions containing the microtubule-associated protein tau) and
genetic risk factors (such as apolipoprotein E).
Both conditions were originally described around the turn of the last century but
languished without significant research effort for decades. In the 1980s, breakthroughs in
pathobiochemistry and genetics led to identification of molecular players in these diseases,
enabling a very fruitful period of biomedical research that continues to intensify. Recent
years have seen a growing interest in the neurobiology of neuronal dysfunction in these
conditions with increasing application of complex techniques from molecular and cellular
neuroscience. Thus, the diversity and sophistication of methods and protocols used for
research on AD and FTD continue to grow. It is not uncommon, and actually is expected
in many journals, to see publications that include techniques as divergent in their required
expertise as behavior, electrophysiology, confocal microscopy, and hardcore biochemistry.
Consequently, projects in AD and FTD research may require individual investigators to
branch out into complex approaches for which they have not received abundant hands-on
training. The goal of this book is to make many of those techniques more accessible.
The book is intended for scientists of all kinds studying AD and FTD. Realizing that
many of the approaches will be foreign to some users, the protocols are presented in a
step-by-step fashion with complete materials lists and user notes describing the “real story”
about how to make the method work.
The book begins with an overview of the two diseases and modern approaches to
research on them. Many of the molecules associated with AD and FTD are notoriously
difficult to work with, so the first half of the book (Chaps. 2–10) details specialized proto-
cols for working with amyloid-b peptide, tau, and apolipoprotein E. The second part
(Chaps. 11–18) focuses on experimental systems for studying AD and FTD, including cell
and animal models, and outcome measures that can be used to assess neuronal function in
these systems.
Birmingham AL, USA Erik D. Roberson
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Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Contemporary Approaches to Alzheimer’s Disease
and Frontotemporal Dementia ....................................... 1
Erik D. Roberson
Part I WorkIng WIth aD- anD FtD-relateD Molecules
2 Preparing Synthetic Ab in Different Aggregation States..................... 13
W. Blaine Stine, Lisa Jungbauer, Chunjiang Yu, and Mary Jo LaDu
3 Isolation of Low-n Amyloid b-Protein Oligomers
from Cultured Cells, CSF, and Brain................................... 33
Ganesh M. Shankar, Alfred T. Welzel, Jessica M. McDonald,
Dennis J. Selkoe, and Dominic M. Walsh
4 Detecting Ab*56 Oligomers in Brain Tissues ............................ 45
Mathew A. Sherman and Sylvain E. Lesné
5 Assessing Ab Aggregation State by Atomic Force Microscopy................ 57
Justin Legleiter
6 Measuring APP Carboxy-Terminal Fragments............................ 71
Luke A. Esposito
7 Detection of APP Intracellular Domain in Brain Tissue..................... 85
Sanjay W. Pimplikar and Anupama Suryanarayana
8 Cell-Based Assays for Regulators of Tau Biology.......................... 93
Umesh K. Jinwal and Chad A. Dickey
9 Split GFP Complementation Assay for Quantitative Measurement
of Tau Aggregation In Situ.......................................... 109
Wanjoo Chun, Geoffrey S. Waldo, and Gail V.W. Johnson
10 Apolipoprotein E Expression and Purification............................ 125
Yvonne Newhouse and Karl H. Weisgraber
Part II MoDel systeMs anD outcoMe Measures
11 Ab Toxicity in Primary Cultured Neurons............................... 141
Adriana Ferreira, Roxana C. Sinjoanu, Alexandra Nicholson,
and Sara Kleinschmidt
12 Manipulation of Gene Expression in the Central Nervous System
with Lentiviral Vectors ............................................. 155
Binggui Sun and Li Gan
vii
viii Contents
13 Selecting a Mouse Model of Alzheimer’s Disease ......................... 169
Jeannie Chin
14 Monitoring Spatial Learning and Memory in Alzheimer’s Disease
Mouse Models Using the Morris Water Maze............................ 191
Kimberly Scearce-Levie
15 Step-by-Step In Situ Hybridization Method for Localizing Gene
Expression Changes in the Brain...................................... 207
Jorge J. Palop, Erik D. Roberson, and Inma Cobos
16 Real-Time Visualization of Axonal Transport in Neurons ................... 231
Yasuko Osakada and Bianxiao Cui
17 Quantifying Biomarkers of Cognitive Dysfunction and Neuronal
Network Hyperexcitability in Mouse Models of Alzheimer’s Disease:
Depletion of Calcium-Dependent Proteins and Inhibitory
Hippocampal Remodeling .......................................... 245
Jorge J. Palop, Lennart Mucke, and Erik D. Roberson
18 Epigenetic Changes in the Brain: Measuring Global Histone Modifications...... 263
Gavin Rumbaugh and Courtney A. Miller
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Contributors
JeannIe chIn • Department of Neuroscience and Farber Institute for Neurosciences,
Thomas Jefferson University, Philadelphia, PA, USA
WanJoo chun • Department of Pharmacology, College of Medicine,
Kangwon National University, Chunchon, Korea
InMa cobos • Nina Ireland Laboratory of Developmental Neurobiology, Department
of Psychiatry, University of California, San Francisco, San Francisco, CA, USA
bIanxIao cuI • Department of Chemistry, Stanford University, Stanford, CA, USA
chaD a. DIckey • Department of Molecular Medicine, Byrd Alzheimer’s Institute,
University of South Florida, Tampa, FL, USA
luke a. esPosIto • ProteoTech, Inc, Kirkland, WA, USA
aDrIana FerreIra • Department of Cell and Molecular Biology,
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
lI gan • Department of Neurology, Gladstone Institute of Neurological Disease,
University of California, San Francisco, San Francisco, CA, USA
uMesh k. JInWal • Department of Molecular Medicine, Byrd Alzheimer’s Institute,
University of South Florida, Tampa, FL, USA
gaIl V.W. Johnson • Department of Anesthesiology, University of Rochester Medical
Center, Rochester, NY, USA
lIsa Jungbauer • Department of Anatomy and Cell Biology,
University of Illinois at Chicago, Chicago, IL, USA
sara kleInschMIDt • Department of Cell and Molecular Biology,
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Mary Jo laDu • Department of Anatomy and Cell Biology,
University of Illinois at Chicago, Chicago, IL, USA
JustIn legleIter • The C . Eugene Bennett Department of Chemistry,
West Virginia University, Morgantown, WV, USA
sylVaIn e. lesné • Department of Neuroscience, Institute for Translational
Neuroscience, University of Minnesota, Minneapolis, MN, USA
JessIca M. McDonalD • Laboratory for Neurodegenerative Research,
The Conway Institute for Biomolecular and Biomedical Research,
University College Dublin, Belfield Dublin, Ireland
courtney a. MIller • Department of Metabolism & Aging and Department
of Neuroscience, The Scripps Research Institute Florida, Jupiter, FL, USA
lennart Mucke • Gladstone Institute of Neurological Disease,
University of California, San Francisco, San Francisco, CA, USA
yVonne neWhouse • Gladstone Institute of Neurological Disease,
University of California, San Francisco, San Francisco, CA, USA
alexanDra nIcholson • Department of Cell and Molecular Biology,
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
ix
x Contributors
yasuko osakaDa • Department of Chemistry, Stanford University,
Stanford, CA, USA
Jorge J. PaloP • Department of Neurology, Gladstone Institute of Neurological
Disease, University of California, San Francisco, San Francisco, CA, USA
sanJay W. PIMPlIkar • Department of Neurosciences, Lerner Research Institute,
Cleveland Clinic, Cleveland, OH, USA
erIk D. roberson • Departments of Neurology and Neurobiology,
Center for Neurodegeneration and Experimental Therapeutics,
University of Alabama at Birmingham, Birmingham, AL, USA
gaVIn ruMbaugh • Department of Neuroscience, The Scripps Research Institute
Florida, Jupiter, FL, USA
kIMberly scearce-leVIe • Genentech, Inc, South San Francisco, CA, USA
DennIs J. selkoe • Center for Neurologic Diseases, Brigham & Women’s Hospital
and Harvard Medical School, Boston, MA, USA
ganesh M. shankar • Center for Neurologic Diseases, Brigham & Women’s Hospital
and Harvard Medical School, Boston, MA, USA
MatheW a. sherMan • Department of Neuroscience, Institute for Translational
Neuroscience, University of Minnesota, Minneapolis, MN, USA
roxana c. sInJoanu • Department of Cell and Molecular Biology,
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
W. blaIne stIne • Department of Anatomy and Cell Biology, University of Illinois at
Chicago, Chicago, IL, USA
bIngguI sun • Department of Neurology, Gladstone Institute of Neurological Disease,
University of California, San Francisco, San Francisco, CA, USA
anuPaMa suryanarayana • Department of Neurosciences, Lerner Research Institute,
Cleveland Clinic, Cleveland, OH, USA
geoFFrey s. WalDo • Bioscience Division, Los Alamos National Laboratory,
Los Alamos, NM, USA
DoMInIc M. Walsh • Laboratory for Neurodegenerative Research,
The Conway Institute for Biomolecular and Biomedical Research,
University College Dublin, Belfield Dublin, Ireland
karl h. WeIsgraber • Gladstone Institute of Neurological Disease,
University of California, San Francisco, San Francisco, CA, USA
alFreD t. Welzel • Laboratory for Neurodegenerative Research,
The Conway Institute for Biomolecular and Biomedical Research,
University College Dublin, Belfield Dublin, Ireland
chunJIang yu • Department of Anatomy and Cell Biology,
University of Illinois at Chicago, Chicago, IL, USA
Chapter 8
Cell-Based Assays for Regulators of Tau Biology
Umesh K. Jinwal and Chad A. Dickey
Abstract
Understanding the molecular mechanisms of Alzheimer’s disease (AD) is a challenging endeavor, namely,
due to the fact that the disease only occurs in the central nervous system of elderly humans. Thus, model
systems simply do not accurately portray this cellular landscape. While we cannot ask many mechanistic
questions using the human brain as our test subject, cell culture techniques that have emerged at least
provide us with the ability to pose fundamental biochemical questions, which may ultimately lead to
translational outcomes for AD. In particular, the intracellular microtubule-associated protein tau that
accumulates in AD is found in normal cells. Manipulating these cells may allow us to address basic ques-
tions about tau biology that would provide novel therapeutic strategies down the road. Here, we describe
several techniques to explore tau cell biology using the Odyssey® Infrared Imaging system (Odyssey sys-
tem) from LI-COR Biosciences. We provide a detailed protocol on how to perform a scalable drug
screening assay called the In-Cell Western and follow-up these screens with standard Western analysis to
confirm whether “hits” are valid by more traditional means. We provide some tips on where mistakes are
most likely to occur, and we interpret our standard Western data, providing some estimation as to the
composition of the banding pattern that is typical for this enigmatic protein.
Key words: Odyssey, Tau, In-Cell Western, Near-infrared, Fluorescence, Cell culture
1. Introduction
For most neurodegenerative protein aggregation disorders, intra-
cellular accumulation of an aberrant form of the parent protein
initiates the cascade that ultimately culminates in the presentation
of a pathological marker detected by histochemistry in post-mor-
tem tissue. While these postmortem analyses have been critical to
establish the foundation of these diseases, it is the early events
that seed the pathology that will likely be the best targets for
therapeutic design. The microtubule-associated protein tau
accumulates into tangles in Alzheimer’s disease (AD), although
the pathogenic process leading up to this is still largely unclear.
Erik D. Roberson (ed.), Alzheimer’s Disease and Frontotemporal Dementia, Methods in Molecular Biology, vol. 670,
DOI 10.1007/978-1-60761-744-0_8, © Springer Science+Business Media, LLC 2011
93
94 Jinwal and Dickey
While mouse modeling has furthered our understanding of the
disease process, these studies carry a high cost, both in time and
resources. Therefore, the emergence of cell culture models has
been critical for understanding the mechanisms at play in tau
pathogenicity. With these models, we have been able to take
advantage of a number of emerging technologies that are helping
us define our understanding of the mechanisms involved with tau
biology (1–3). Moreover, we are continuing to discover thera-
peutic targets that might prove most effective in our fight against
AD and other tauopathies. In this chapter, we describe the meth-
ods using the Odyssey® Infrared Imaging system from LI-COR
Biosciences, which has allowed us to begin to address questions
in a semi-high throughput quantitative format previously not
achievable for the tau protein. Screening the effects of small mol-
ecules, siRNAs and even biological agents, such as growth factors
and cytokines, can be done in a high volume within a two-day
protocol. The dual-color capability provides an option for greater
control and reliability of the assay in a single run. Herein, we
provide basic protocols to analyze total tau levels relative to
GAPDH; however, this technique can be applied to any number
of intracellular targets, provided antibody specificity is high. We
also describe validation studies using the Odyssey system for more
traditional Western blotting.
1.1. In-Cell Western The LI-COR Biosciences In-Cell Western (ICW) assay is suited
for drug discovery assay development where protein quantifica-
tion is important in the cellular environment. It incorporates the
use of near infrared-labeled secondary antibodies, which can be
independently detected with the dual laser Odyssey system at
680 nm and 800 nm, to detect any combination of two proteins,
provided species-specific antibodies are available. The assay can
quantify proteins within a monolayer of fixed cells in 96 or 384-
well microplates, making this platform a cross between high-
throughput immunofluorescent chemistry and Western blotting;
however, with this increased throughput, resolution is lost, mak-
ing follow-up validation studies critical. This procedure is unique
from typical plate reader-based applications and other biological
system applications, such as the Typhoon from GE, because of
the use of near-infrared laser scanning. Near-infrared light has a
longer wavelength than light in the visible range. Therefore, near-
infrared light can travel farther and pass through certain surfaces
without losing intensity. Thus, the Odyssey system is the most
affordable of a handful of devices that allows for this type of appli-
cation. The signal from the Odyssey system is linear and, thus, the
values provided by the system software allow for relative quantita-
tion within each plate scanned. Moreover, a normalization tem-
plate can be used for variance across plates, allowing for multiplate
assay analyses. Here, we describe a protocol specific for analyzing